INK4a/ARF encodes the ARF tumor suppressor protein and is the second most frequently inactivated gene in human cancers. ARF prevents oncogene-mediated transformation through protein-protein interactions in partially defined p53-dependent and p53-independent pathways. Numerous ARF-associated proteins have been identified but few besides the p53 antagonist, Mdm2, have a clearly defined role in ARF-mediated tumor suppression. Based on work from the previously funded project, we identified and characterized two novel ARF interacting proteins and showed they participate in ARF signaling. Parf (Partner of ARF) is required for ARF's p53-independent growth suppressive activity, whereas NIAM (Nuclear Interactor of ARF and Mdm2) activates p53 and collaborates with ARF to inhibit cell proliferation. Both proteins also act independent of ARF and p53 to maintain genomic stability, regulate DNA damage checkpoints, and suppress cancer cell proliferation, suggesting they have anticancer activities that intersect with ARF and other antiproliferative pathways. However, the underlying mechanisms by which Parf and NIAM regulate ARF signaling, genomic stability and cell proliferation are poorly understood. The specific objective of this research is to define the mechanistic and biological roles of these novel ARF interacting proteins in ARF signaling and carcinogenesis. Aim 1 will establish how NIAM enhances ARF signaling and its role in tumorigenesis. Aim 2 will define how Parf contributes to ARF signaling and tumor suppression. Molecular and biochemical approaches, as well as unique mouse models of cancer, will be utilized. These studies will advance our fundamental understanding of how the ARF tumor suppressor and its associated proteins prevent cancer. This is highly relevant to public health because loss of ARF figures prominently in the development of nearly all types of human tumors and we cannot effectively battle cancer until we know its molecular basis. This work is expected to identify new targets for anticancer strategies and new paradigms used by tumor suppressors to prevent tumorigenesis, and as such, it should improve our ability to diagnose, treat and ultimately prevent human cancer.